The detection and quantification of nucleic acid sequences is of importance for a wide range of applications. The most widely used method to detect nucleic acids are based on the polymerase chain reaction (PCR). PCR is used to amplify a segment of DNA flanked by stretches of known sequences. Two oligonucleotides binding to these known flanking sequences are used as primers for a series of in vitro reactions that are catalyzed by a DNA polymerase. These oligonucleotides typically have different sequences and are complementary to sequences that lie on opposite strands of the template DNA and flank the segment of DNA that is to be amplified. The template DNA is first denatured by heat in the presence of a large molar excess of each of the two oligonucleotides and the four 2′-deoxynucleotide triphosphates. The reaction mixture is then cooled to a temperature that allows the oligonucleotide primers to anneal to their target sequences. Afterwards, the annealed primers are extended by the DNA polymerase. The cycle of denaturation, annealing, and DNA-synthesis is then repeated about 10 to 50 times. Since the products of one cycle are used as a template for the next cycle the amount of the amplified DNA fragment is theoretically doubled with each cycle resulting in a PCR-efficiency of 100%.
“Real-time PCR” refers to a polymerase chain reaction that is monitored, usually by fluorescence, over time during the amplification process, to measure a parameter related to the extent of amplification of a particular sequence, such as the extent of hybridization of a probe to amplified target sequences. The DNA generated within a PCR is detected on a cycle by cycle basis during the PCR reaction. The amount of DNA increases faster the more template sequences are present in the original sample. When enough amplification products are made a threshold is reached at which the PCR products are detected. Thus amplification and detection are performed simultaneously in the same tube.
In biological research, PCR has accelerated the study of testing for communicable diseases. Medical applications of PCR include identifying viruses, bacteria and cancerous cells in human tissues. PCR can even be used within single cells, in a procedure called in situ (in-site) PCR, to identify specific cell types. PCR can also be applied to the amplification of RNA, a process referred to as reverse transcriptase PCR (RT-PCR). RT-PCR is similar to regular PCR, with the addition of an initial step in which DNA is synthesized from the RNA target using an enzyme called a reverse transcriptase. A wide variety of RNA molecules have been used in RT-PCR, including ribosomal RNA, messenger RNA and genomic viral RNA.
PCR itself is quite simple, but sample preparation can be laborious. The goals of sample preparation include the release of nucleic acid (DNA or RNA), concentration of the nucleic acid to a small volume for PCR, and removal of inhibitors of PCR. Inhibitors of PCR are naturally occurring substances which reduce the efficiency of PCR, and which are often present in clinical samples. When the specimen contains a large amount of target nucleic acid, sample preparation is trivial. But sample preparation is more difficult in most clinical specimens, particularly when a large volume specimen must be processed and only a few pathogens are present. Complex protocols are often required.
Since PCR detects the presence or absence of a particular nucleic acid target, it will only detect a pathogen if its nucleic acid is present in the particular specimen. PCR detects nucleic acids from living or dead microbes. This must be recognized if PCR is used to monitor response to therapy. PCR provides at most nucleic acid sequence information. PCR can be used to screen for drug resistance mutations, but it does not provide direct antibiotic susceptibility data.
Appropriate controls are necessary when PCR is used diagnostically. These include negative controls, positive controls and specificity controls. Negative controls (no target DNA) are needed to detect contamination. Contamination can occur during sample preparation or reagent mixing, so negative controls need to be processed in parallel with clinical samples. Negative controls should be interspersed among the samples to detect cross-contamination from sample to sample. Contamination is frequently intermittent; a sufficient number of negative controls must be included to detect low rates of contamination. Most published studies have not included a sufficient number of negative controls.
Positive controls include a small number of target DNA copies. Positive controls are needed to ensure efficient release of target DNA from pathogens, to guard against loss of DNA during sample processing, and to identify the presence of inhibitors (natural substances sometimes present in clinical samples that reduce PCR efficiency). Positive controls should be processed in parallel with clinical specimens. Clinical specimens vary in the presence of inhibitors of PCR, and it may be necessary to add an internal positive control for each sample. The internal positive controls have the same recognition sites as the target DNA, but are designed with some difference in the internal sequence. Amplification of the internal positive controls can be distinguished from that of the real target DNA.
Specificity controls are needed to determine the range of target DNAs that will be amplified by the PCR assay. For assays designed to detect pathogens in clinical samples, human DNA samples must be tested to ensure that the PCR primers do not recognize a human DNA target by chance. Related pathogens must be tested to determine the range of species/strains that will be amplified. Specificity controls are needed only once, when a new PCR assay is designed. Negative and positive controls must be included every time samples are processed, and should be processed simultaneously with the clinical samples.
PCR has been used in three broad categories of diagnostic procedures, namely detection, characterization and quantification.
Detection is the most difficult PCR procedure, especially when the number of pathogens in the specimen is low. The PCR must be conducted under conditions of high sensitivity. Many temperature cycles are used, or a nested protocol is used in which the products from the first reaction are re-amplified with a second set of primers. This makes PCR for detection especially prone to carryover contamination. Sample preparation may be laborious, as there is an attempt to process as large a specimen volume as possible. Inhibitors of PCR occur naturally in many clinical samples, and are a major limitation. Numerous positive and negative controls must be included as described above.
In a characterization procedure, nucleic acid variants are identified based on the nucleic acid sequence between the two PCR primers. Many techniques can be used to detect variable sequences, including length polymorphism, changes in restriction sites, and direct DNA sequencing. This is often the easiest type of PCR to carry out clinically. Ample quantities of nucleic acid target can be present in the specimen, either an already grown bacterial or viral culture or a clinical sample with large numbers of microbes. Goals can include rapid detection of drug resistance mutations, assignment of strains to clinically meaningful phylogenetic groups, or epidemiological tracing.
Quantitation (indicating how many copies of the target nucleic acid are present) has primarily been applied to chronic viral infections, especially hepatitis C virus (HCV) and human immunodeficiency virus (HIV) infections. The level of viremia has prognostic implications, and has been used to demonstrate response to antiviral drugs. PCR is quite sensitive, but it is not inherently quantitative. The amount of the final PCR product is usually similar from an initial sample containing 10 or 10,000 copies. This limitation can be overcome by serial dilution of the clinical sample until no target DNA is detected, or by the addition of synthetic competitor DNA molecules. The competitor molecules have regions complementary to the two primers, but differ in some way from the natural target (e.g., a different length). By comparing the amount of the natural and competitor PCR products, a rough estimation of the number of target molecules in the sample is possible.
PCR has been applied in the research setting to hundreds of pathogens, and has yielded important insights into pathogenesis and epidemiology of many infectious diseases. For clinical purposes, PCR-based diagnostic tests are best applied when the following conditions are fulfilled: (1) The results of the test will make a clear clinical difference and a therapy will be given or withheld based on the results of PCR; (2) routine culture methods are limited because the microbe cannot be grown (e.g., Mycobacterium leprae, HCV), grows slowly (e.g., M. tuberculosis), or is difficult to culture (e.g., Brucella species, HIV); and (3) there is an accessible clinical specimen which contains large numbers of microbes (e.g., blood for HCV or HIV).
PCR has been useful in a variety of chronic virus infections (HIV, HCV, hepatitis B virus, human papillomavirus and cytomegalovirus). PCR has been crucial for the detection of HIV infection in neonates, since maternal antibodies complicate serologic diagnosis. Quantitation of HIV and HCV viremia by PCR has important prognostic implications, and has been used to monitor response to drug therapy. PCR is useful for the rapid diagnosis of pulmonary infections in immuno-compromised hosts, particularly for cytomegalovirus and Pneumocystis carinii. 
HIV
The human immunodeficiency virus type-1 (HIV-1) is a retrovirus belonging to the family of the Lentiviridae. One of the characteristic features of this virus group is that the members replicate over a DNA intermediate through the viral encoded reverse transcriptase (RT) enzyme activity. The high replication rate combined with the low fidelity of that reverse transcriptase enzyme provides the virus with an extremely high genomic flexibility. As a consequence, different levels of genetic variability are observed for HIV-1. The epidemic is characterized by the presence of clades within the M-group virus, but there is also an O-group and an N-group virus described, each of them again harboring a variety of clades. Quasispecies populations within the infected individual are also seen. Clinically, there are some important consequences to this quasispecies concept, for example, in vaccine development and immune escape. This concept contributes to the emergence of drug resistant variants that surface under antiviral treatments.
In order to control the course of the disease in infected individuals, potent highly active anti-retroviral therapies (HAART) have been designed. Due to the ongoing replication of the virus, anti-retroviral drug resistance eventually develops, leading to therapy failure. Therefore, there is an ongoing need for more and more potent anti-HIV-1 drugs.
To assess the efficacy of drugs in the treatment of patients in vivo, clinical markers of virus replication needed to be defined. In the past, some surrogate markers, like CD-cell count, have been used. More recently, some commercial assays like Quantiplex (Chiron), NucliSense (Organon-Teknika) and Amplicor HIV-1 Monitor (Roche) were developed to directly measure viral load. These viral load determinations proved to be an excellent tool in monitoring therapeutic efficiency for HAART and for clinical trials with new experimental drugs.
The design of an HIV-1 viral load test is a real challenge. Ideally, a viral load test should fulfill to the following criteria:                i) be able to detect the huge variability of clades within one group with the same efficiency;        ii) have a dynamic range of at least five logs or higher; and        iii) the lower limit of detection should be as low as a few viral copies/mL. Although variability at the PCR-primer binding sites is a real concern in assay development, RT-PCR based assays are considered as the most sensitive technologies.Mitochondrial Toxicity        
Mitochondrial toxicity is clearly recognized as an adverse effect of long-term use of antiviral agents, in particular reverse transcriptase inhibitors. Clinical features of this mitochondrial toxicity vary depending on the tissues that are affected. It is largely dependent on the aerobic metabolism needed for energy supply required for that particular tissue. Most toxic events are reversible at an early stage, however lactic acidosis is often irreversible and can result in death.
The common pathway of antiviral agent induced toxicity is mitochondrial dysfunction. The antiviral agent (most likely the triphosphate form of a nucleoside analogue) inhibits the mitochondrial DNA polymerase γ leading to the loss of mitochondria. This enzyme is essential for the replication of the mitochondrial genome. Tissues with high ATP demand are most susceptible to mitochondrial toxicity.
The mechanism underlying this mitochondrial dysfunction includes failure of energy dependent ionic balance. Subsequently, there is an increase in intracellular calcium, initiating lipolysis and proteolysis, and leading to the accumulation of lactic acid and partial reduction of the respiratory activities.
Since the mitochondrial dysfunction develops over months and symptoms are initially mild, it is important to develop sensitive diagnostic tests that allow determination of the enzyme activity and inhibition by the selected antiviral agent. Evenly important, new candidate antiviral agents need to be evaluated for their unfavorable DNA polymerase γ inhibiting capacities.
Hepatitis C
Hepatitis C virus (HCV) infection is a pandemic infection, and is a major cause of liver disease. Reports of successful treatment of HCV infection with interferon have increased interest in applications of RT-PCR.
Available tests for HCV infection are limited. Initial serologic tests for HCV had poor sensitivity. Second and third-generation serologic tests have improved sensitivity, but are still not completely dependable. HCV RNA is readily detected in serum using RT-PCR. Viremic patients typically have very high viral titers.
PCR has been applied to the diagnosis of HCV infection in a variety of clinical settings. HCV can be detected as early as one week after infection, and PCR can be used to detect HCV infection during the “window” period between infection and seroconversion. HCV PCR is useful for detecting HCV in seronegative individuals with liver disease. It can be used to confirm maternal to fetal spread of HCV. HCV PCR may be useful in the evaluation of seropositive individuals as candidates for interferon or other therapies. Portions of HCV-seropositive patients are negative by HCV PCR, and may have resolved their infections. PCR-negative individuals have lower serum transaminase concentrations and less histologic activity on liver biopsies. Long-term follow-up studies are needed, but it may be reasonable to withhold therapy from patients with negative HCV PCR results.
The amount of HCV viremia can be determined by either quantitative PCR. PCR is sensitive and is quantitative over a wide range of viral titers. High-titer viremia is correlated with an advanced disease stage. The prognostic value of HCV quantitation awaits prospective studies, but the level of viremia may be useful in selecting candidates for therapy. Quantitative HCV PCR also appears to be useful in monitoring the response to therapy.
WO 00/44936 filed by Bavarian Nordic Research Institute A/S describes a real-time PCR method for the detection and quantification of variants of nucleic acid sequences which differ in the probe-binding site. The method is based in the complete or partial amplification of the same region of the variants and the addition of two or more oligonucleoitde probes to the same PCR mixture, each probe being specific for the probe-binding site of at least one variant.
WO 01/66799 filed by E.I. DuPont Nemours and Company discloses a PCR-based dsDNA quantification method that monitors the fluorescence of a target, whose melting characteristics is predetermined, during each amplification cycle at selected time points. By selecting targets with distinguishing melting curve characteristics, multiple targets can be simultaneously detected.
WO 00/68436 filed by Nationales Zentrum fur Retroviren discloses sequences allowing the detection and quantification of human immunodeficiency virus.
U.S. Pat. No. 6,235,504 assigned to the Rockefeller University describes methods for identifying genetic sequences useful as genomic equivalent markers for organisms.
U.S. Pat. No. 6,210,875 discloses a process for determining the efficacy of antiviral therapy in an HIV-infected host that includes detecting the level of transcriptionally active HIV in the monocytes of the subject at a plurality of times by simultaneously exposing the monocytes to an oligonucleotide probe that specifically binds to at least a portion of HIV mRNA and exposing the monocytes to an antibody, wherein the oligonucleotide probe is labeled with a fluorescent label, comparing the detected HIV levels, and correlating the HIV levels over time with the therapy regimen.
U.S. Pat. No. 5,843,640 discloses an in situ process of simultaneously detecting a specific predetermined nucleic acid sequence and a specific predetermined cellular antigen in the same cell.
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Cancer Res 1998;58:3957-3964; Gelmini S, Orlando C, Sestini R, et al. Quantitative polymerase chain reaction-based homogeneous assay with fluorogenic probes to measure c-erB-2 oncogene amplification. Clin Chem 1997;43:752-758; deKok J B, Hendriks J C M, van Solinge W W, Willems H L, Mensink E J, Swinkels D W. Use of real-time quantitative PCR to compare DNA isolation methods. Clin Chem 1998;44:2201-2204; Lockey C, Otto E, Long Z. Real-time fluorescence detection of a single DNA molecule. Biotechniques 1998;24:744-746; Marcucci G, Livak K J, Bi W, Strout M P, Bloomfield C D, Caligiuri M A. Detection of minimal residual disease in patients with AML1/ETO-associated acute myeloid leukemia using a novel quantitative reverse transcription polymerase chain reaction assay. Leukemia 1998;12:1482-1489; Suryanarayana K, Wiltrout T A, Vasquez G M, Hirsch V M, Lifson J D. Plasma SIV RNA viral load determination by real-time quantification of product generation in reverse transcriptase-polymerase chain reaction. AIDS Res Hum Retroviruses 1998;14:183-189; Morris T, Robertson B, Gallagher M. Rapid reverse transcription-PCR detection of hepatitis C virus RNA in serum by using the TaqMan fluorogenic detection system. J Clin Microbiol 1996;34:2933-2936; Swan D C, Tucker R A, Holloway B P, Icenogle J P. A sensitive, type-specific, fluorogenic probe assay for detection of human papillomavirus DNA. J Clin Microbiol 1997;35:886-891; McGoldrick A, Lowings J P, Ibata G, Sands J J, Belak S, Paton DJ. A novel approach to the detection of classical swine fever virus by RT-PCR with a fluorogenic probe (TaqMan). J Virol Methods 1998;72:125-135; Abe, A., K. Inoue, T. Tanaka, J. Kato, N. Kajiyama, R. Kawaguchi, S. Tanaka, M. Yoshiba, and M. Kohara 1999. Quantitation of hepatitis B virus genomic DNA by real-time detection PCR. J Clin Microbiol. 37:2899-2903; Aberham, C., C. Pendl, P. Gross, G. Zerlauth, and M. Gessner 2001. A quantitative, internally controlled real-time PCR Assay for the detection of parvovirus B19 DNA. J Virol Methods. 92:183-191; Bisset, L. R., S. Bosbach, Z. Tomasik, H. Lutz, J. Schupbach, and J. Boni 2001. Quantification of in vitro retroviral replication using a one-tube real-time RT-PCR system incorporating direct RNA preparation, J Virol Methods. 91:149-155; Cane, P. A., P. Cook, D. Ratcliffe, D. Mutimer, and D. Pillay 1999. Use of real-time PCR and fluorimetry to detect lamivudine resistance-associated mutations in hepatitis B virus. Antimicrob Agents Chemother. 43:1600-1608; Cubic, H. A., A. L. Seagar, E. McGoogan, J. Whitehead, A. Brass, M. J. Arends, and M. W. Whitley 2001. Rapid real time PCR to distinguish between high risk human papillomavirus types 16 and 18. Mol. Pathol. 54:24-29; Desire, N., A. Dehee, V. Schneider, C. Jacomet, C. Goujon, P. M. Girard, W. Rozenbaum, and J. C. Nicolas 2001. Quantification of human immunodeficiency virus type 1 proviral load by a TaqMan real-time PCR assay. J Clin Microbiol. 39:1303-1310; Gault, E., Y. Michel, A. Dehee, C. Belabani, J. C. Nicolas, and A. Garbarg-Chenon 2001. Quantification of human cytomegalovirus DNA by real-time PCR. J Clin Microbiol. 39:772-775; Gniber, F., F. G. Falkner, F. Dorner, and T. Harmnerle 2001. Quantitation of viral DNA by real-time PCR applying duplex amplification, internal standardization, and two-color fluorescence detection. Appl Environ Microbiol. 67:2837-2839; Jabs, W. J., H. Hennig, M. Kittel, K. Pethig, F. Smets, P. Bucsky, H. Kirchner, and H. J. Wagner 2001. Normalized quantification by real-time PCR of Epstein-Barr virus load in patients at risk for posttransplant lymphoproliferative disorders. 3 Clin Microbiol. 39:564-569; Josefsson, A., K. Livak, and U. Gyllensten 1999. Detection and quantitation of human papillomavirus by using the fluorescent 5′ exonuclease assay. J Clin Microbiol. 37:490-496; Kato, T., M. Mizokami, M. Mukaide, E. Orito, T. Ohno, T. Nakano, Y. Tanaka, H. Kato, F. Sugauchi, R. Ueda, N. Hirashima, K. Shimamatsu, M. Kage, and M. Kojiro 2000. Development of a TT virus DNA quantification system using real-time detection PCR. J Clin Microbiol. 38:94-98; Kearns, A. M., M. Guiver, V. James, and J. King 2001. Development and evaluation of a real-time quantitative PCR for the detection of human cytomegalovirus. J Virol Methods. 95:121-131; Kessler, H. H., G. Muhlbauer, B. Rinner, E. Stelzl, A. Berger, H. W. Dorr, B. Santner, E. Marth, and H. Rabenau 2000. Detection of Herpes simplex virus DNA by real-time PCR. J Clin Microbiol. 38:2638-2642; Kimura, H., M. Morita, Y. Yabuta, K. Kuzushima, K. Kato, S. Kojima, T. Matsuyama, and T. Morishima 1999. Quantitative analysis of Epstein-Barr virus load by using a real-time PCR assay. J Clin Microbiol. 37:132-136; Komurian-Pradel, F., G. Paranhos-Baccala, M. Sodoyer, P. Chevallier, B. Mandrand, V. Lotteau, and P. Andre 2001. Quantitation of HCV RNA using real-time PCR and fluorimetry. J Virol Methods. 95:111-119; Kuimelis, R. G., K. J. Livak, B. Mullah, and A. Andrus 1997. Structural analogues of TaqMan probes for real-time quantitative PCR. Nucleic Acids Symp Ser. 37:255-256; Lallemand, F., N. Desire, W. Rozenbaum, J. C. Nicolas, and V. Marechal 2000. Quantitative analysis of human herpesvirus 8 viral load using a real-time PCR assay. J Clin Microbiol. 38:1404-1408; Lewin, S. R., M. Vesanen, L. Kostrikis, A. Hurley, M. Duran, L. Zhang, D. D. Ho, and M. Markowitz 1999. Use of real-time PCR and molecular beacons to detect virus replication in human immunodeficiency virus type 1-infected individuals on prolonged effective antiretroviral therapy. J. Virol. 73:6099-6103. Locatelli, G., F. Santoro, F. Veglia, A. Gobbi, P. Lusso, and M. S. Malnati 2000. Real-time quantitative PCR for human herpesvirus 6 DNA. J Clin Microbiol. 37:4042-4048; Machida, U., M. Kami, T. Fukui, Y. 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Development of a real-time quantitative assay for detection of epstein-barr virus. J Clin Microbiol. 38:712-715; Nitsche, A., N. Steuer, C. A. Schrnidt, O. Landt, H. Ellerbrok, G. Pauli, and W. Siegert 2000. Detection of human cytomegalovirus DNA by real-time quantitative PCR. J Clin Microbiol. 38:2734-2737; Obyashiki, J. H., A. Suzuki, K. Aritaki, A. Nagate, N. Shoji, K. Ohyashiki, T. Ojima, K. Abe, and K. Yamamoto 2000. Use of real-time PCR to monitor human herpesvirus 6 reactivation after allogeneic bone marrow transplantation. Int J Mol Med. 6:427-432; Pevenstein, S. R., R. K. Williams, D. McChesney, E. K. Mont, J. E. Smialek, and S. E. Straus 1999. Quantitation of latent varicella-zoster virus and herpes simplex virus genomes in human trigeminal ganglia. J. Virol. 73:10514-10548; Ratge, D., B. Scheiblhuber, M. Nitsche, and C. Knabbe 2000. High-speed detection of blood-borne hepatitis C virus RNA by single-tube real-time fluorescence reverse transcription-PCR with the LightCycler. 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Although assays exist for the diagnosis and evaluation of viral infections, additional assays and kits are needed that provide a more sensitive or precise analysis of the condition of a diseased cell. More sensitive and precise methods are also needed to assess the activity of a compound or substance against a target virus and to assess host toxicity induced by the compound or substance.
It is therefore an object of the present invention to provide a process for the identification of active compounds for the treatment of viral infections.
It is another object of the present invention to provide a process to measure mitochondrial toxicity.
It is another object of the present invention to provide a process for the detection and analysis of viral infections.
It is a further object of the invention to provide a process for the detection and analysis of mitochondrial toxicity.